Type 2 diabetes mellitus (T2DM) and coronary artery disease (CAD) are leading causes of morbidity and mortality worldwide. Development of new and more effective approaches to prevention and treatment requires improved understanding of disease mechanisms. Genetic mapping in humans offers an approach to identify genes and DNA variants underlying the inherited contribution to disease susceptibility, unbiased by prior assumptions about the pathophysiological processes responsible. Next-generation sequencing technologies make it possible for the first time to catalog mutations observed in patients and in healthy controls. While human genetics has the potential to dramatically expand our knowledge of biological and disease mechanisms, progress is constrained by two central challenges: (a) determining which DNA changes are functional, and which are benign, and (b) developing cellular assays with which to interrogate the functions of the genes and variants thereby identified. Specifically, the field requires methods to functionally screen large number of mutations in high throughput, using assays that faithfully represent the human cell types of interest. This proposal is built on a foundation of human genetics, genome engineering, and stem cell biology, and is focused on the major metabolic diseases of T2DM, dyslipidemia, and CAD. The approach leverages three recent advances: (a) ongoing next-generation sequencing studies identifying candidate disease mutations, (b) development of methodologies to rapidly alter genes of interest in human pluripotent stem cells (hPSCs) using engineered TAL effector nucleases (TALENs), and (c) protocols to differentiate hPSCs into cell populations with characteristics of hepatocytes, adipocytes, and pancreatic beta cells. Our proposal combines two central innovations. First, we propose to develop two novel approaches to engineer the genomes of hPSCs, rapidly and efficiently knocking out the function of candidate genes, and introducing specific mutations observed in patients. This will generate isogenic human stem cells that differ only at a single mutation of interest. Second, we will develop and improve protocols to differentiate hPSCs into physiologically mature hepatocytes, adipocytes, and beta cells. By engineering stem cells to carry specific mutations, and by differentiating these engineered stem cells into physiologically relevant human metabolic cell types, we will make it possible to study the impact of large numbers of gene variants on human cell biology and function. By relating the functions of gene mutations and cell biological processes with the phenotypes of human patients, we will provide pathophysiological insights and practical in vitro assays to guide development of therapeutics for these challenging diseases.